The proposed research concerns the elucidation of regulatory mechanisms involved in the processes of mitochondrial energy generation in liver tissue. Specific emphasis will be assigned to the regulation of the pyruvate and alpha-ketoglutarate dehydrogenase multienzyme complexes of the mitochondrial compartment. Studies will be conducted at the level of the isolated, purified multienzyme complex, the intact mitochondrion, the isolated hepatocyte, and especially the isolated, perfused liver. In the case of the regulation pyruvate dehydrogenase complex an attempt will be made to characterize in intact metabolic systems the contribution to the overall regulation of the enzyme complex of feedback inhibition by the products (NADH and acetyl-CoA) of the active, dephosphoform of the enzyme versus the contribution of regulatory effects exerted by the same molecular species on the kinase-phosphatase mediated interconversion of active and inactive pyruvate dehydrogenase complex. Further, an attempt will be made to characterize the contribution of regulatory effects exerted on the transport of the monocarboxylate substrate, pyruvate. Pyruvate not only serves as the substrate of the multienzyme complex but is a potent inhibitor of the pyruvate dehydrogenase kinase reaction which inactivates the active enzyme. Interesting in this regard will be studies conducted in the perfused liver which will be designed to illustrate possible relationships between the processes of ketogenesis and gluconeogenesis (as well as the hormonal control of these two processes) and the flux of carbon through the pyruvate dehydrogenase reaction. Emphasis will be assigned to the regulatory effects of various nucleotide species (e.g., adenine, guanine and pyrudine and coenzyme A derivatives) on the activity of the alpha-ketoglutarate dehydrogenase complex in various metabolic systems. Especially interesting will be the testing of the thesis that the GTP/GDP ratio of the mitochondrial compartment may be a primary determinant (regulator) of the flux through the gamma-ketoglutarate dehydrogenase step of the tricarboxylic acid cycle. The results of the proposed study should provide a perception of the fine details of a multifactorial system of control of two important enzymatic steps in the energy generating pathways of the liver cell.